Amplification of nucleic acids is widely used in research, diagnostics, forensics, medicine, food science and agriculture. “Point of Care Testing” (POCT) relates to diagnostic testing at or near the site of patient care, i.e. de-centralized examinations that may e.g. be performed in private medical practices, small hospitals, pharmacies, or even in the field or at the site of an accident or other medical emergency or within an ambulance. Point-of-care diagnostics requires rapid and easy testing. Hence, in the case of nucleic-acid based detection methods, fast isothermal amplification technologies have recently become more and more important as compared to the established but slower PCR-based methods. PCR requires thermo-cycling in order to separate the two strands of double-stranded nucleic acids such as DNA. In contrast, isothermal amplification methods for example do not require a thermocycler.
One of the most prominent isothermal amplification methods is the so-called Helicase Dependent Amplification (HDA) (described e.g. in Vincent et al. (2004), EMBO reports 5(8):795-800; Jeong et al. (2009), Cell. Mol. Life. Sci 66:3325-3336; WO-A2 2004/027025; WO-A2 2006/074334; all incorporated by reference herein). HDA is based on the ability of helicases to unwind double-stranded nucleic acids, particularly DNA, without the need for heating or even thermocycling. In HDA, the separated DNA strands are replicated using DNA polymerases and suitable oligonucleotide primers. Hence, HDA mimics the natural replication fork mechanism. HDA requires the presence of ATP, divalent magnesium ions and dNTPs. Some HDA-based methods additionally employ single-stranded binding proteins (SSBs) for the coating of displaced DNA strands. In principal HDA methods can be performed in thermolabile or thermostable reactions depending on the enzymes used. Thermolabile HDA reactions are typically performed at temperatures between 25 and 50° C., preferably between 37 and 42° C. In contrast the thermostable HDA or thermophilic HDA (tHDA) reactions are performed at temperatures above 50° C., typically between 60 and 70° C.
The HDA reaction selectively amplifies a target sequence defined by two primers. HDA uses a helicase enzyme to separate the two strands of double-stranded nucleic acid rather than heat as in PCR. Therefore, HDA can be performed at a single temperature without the need of thermocycling. The steps of a conventional standard HDA reaction are shown in FIG. 1: In the first step a double-stranded nucleic acid is unwound by a helicase resulting in (partially) single-stranded sequences. This is followed by binding of primers to the single-stranded regions. In step 2 a polymerase synthesizes complementary strands. Ultimately, the helicase and the polymerase act together to result in amplification of the template (step 3).
Other isothermal amplification include Strand Displacement Amplification (SDA) which is based on nicking an unmodified strand of DNA using restriction enzymes and extending the 3′ end at the nick through the action of an exonuclease deficient DNA polymerase to displace the downstream DNA strand.
Yet another isothermal amplification is the Rolling Circle Amplification (RCA) in which a linear ssDNA is annealed to a circular ssDNA template which has been generated by joining two ends of the template DNA using a DNA ligase. Subsequently, the annealed primer is extended using a DNA polymerase and tandemly linked copies of the complementary template sequence are generated. RCA and SDA both require an initial heat denaturation step.
Other isothermal amplification include for example the Nicking Enzyme Amplification Reaction (NEAR) and the related Exponential Amplification Reaction (EXPAR) which both employ nicking enzymes and polymerases for the amplification of short double-stranded template sequences (described e.g. in van Ness et al. (2003), Proc. Natl. Acad. Sci. 100(8):4504-4509; US-A1 2003/0082590; WO-A2 2004/022701; WO-A2 2009/012246; WO-A2 2004/067726; all incorporated by reference herein).
However, all of the above-described isothermal amplification methods require complicated reaction protocols, are relatively slow and/or are limited to relatively short template sequences.